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Staci Hammer

Dr. Kate Cooper

Bio 410

18 April 2018

Succinic Acid a Chemoattractant in C. elegans

Abstract

The model organism nematode C. elegans contains the same G-protein coupled receptor (GCPR) ODR-10
as humans. Diacetyl binds to this GPCR, causing nematode migration towards it as a chemoattractant, and
succinic acid’s similar chemical structure and use in humans may also attract these nematodes via ODR-
10. By performing a chemotaxis assay comparing diacetyl and succinic acid, the resulting chemotaxis
indices indicate succinic acid as a chemoattractant. Thus, more research should be invested in this
chemical in relation to GPCRs and receptor specificity.

Introduction

Sensing and responding to the environment is a crucial function for a living organism’s
survival: to find food, avoid predators and ultimate death, and produce future generations by
mating. For the model organism C. elegans nematode, a commonly studied characteristic is its
attraction to diacetyl because it is a byproduct of fermentation, and nematodes eat fermenting
bacteria. Diacetyl is a chemical that binds to ODR-10, a G-protein coupled receptor in C. elegans
and humans (Troemel, Kimmel, & Bergmann, 1997) commonly expressed in AWA olfactory
neurons. In nematodes, these olfactory cues prompt a change in behavior, including attraction or
avoidance of the stimulant, feeding, or mating (Zhang, et. al., 1997). AWA and other types of
neurons relate the appropriate response to multiple chemicals and signals (Bargmann, 2006).

If one neuron can respond to a vast array of chemicals, perhaps two structurally similar
chemicals would elicit the same response if they can also bind to ODR-10. In fact, many
chemicals have been tested to determine whether they alter the behavior of C. elegans
(Bargmann, 2006). However, no chemotaxis assays have been published for the chemical
succinic acid, which has a similar composition to the widely known chemoattractant diacetyl.
Diacetyl’s structure has two acetyl groups linked together while succinic acid contains two
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carbonyl groups as part of the dicarboxylic acid (Fig. 1). Even though diacetyl is a liquid and
succinic acid is a white crystal, there is a possibility the chemicals are similar enough to bind to
the ODR-10 receptor to produce the same response by the nematodes.

Figure 1. The structures of diacetyl (A) and succinic acid (B). Both structures contain carbonyl groups
(C=O) and four carbons, but succinic acid has a hydroxyl group (OH) at both ends.

Moreover, succinic acid is a natural substance in plant and animal tissues and is
commercially used as a food additive for flavoring. It is also commonly incorporated in active
ingredients in pharmaceutical drugs for health benefits (URL 1). Clearly important to humans,
these benefits are probably analogous for C. elegans, and could be another reason they would be
attracted to succinic acid. Knowing whether these nematodes positively respond to this substance
can lead to novel information on the ODR-10 receptor and the specificity of receptors in general.
Thus, the purpose of this experiment is to test whether succinic acid is a chemoattractant like
diacetyl.

Methods

For each of nine fresh agar Petri dish plates, we marked four equal quadrants on the
bottom and created a circle with a radius of 0.5 cm in the center of the plate where each quadrant
met. In each quadrant, we marked a point 2 cm from the inner circle so that each point was the
same distance from each point in the neighboring quadrants, and then each point was
alternatively marked “T” and “C” to get 2 “T’s” and “C’s” per plate.

Then 1mL of M9 buffer was added to a plate of living C. elegans and swirled to collect
the worms. After cutting the tip off a 1mL pipette tip, we collected the M9/worm mixture and
dispensed it into a centrifuge tube. We centrifuged the mixture for 1 minute at 2000 RPM. We
then removed all but 100µL of supernatant. After adding another mL of M9 buffer to the tube,
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we centrifuged the pellet again. This process was repeated three more times to finish with a
pellet and 100µL of buffer in the centrifuge tube.

Next, 3 centrifuge tubes were filled with 25µL of 500mM sodium azide. Then we added
25µL of 1% diacetyl to one of the tubes, 25µL of 100% ethanol to another, and 25µL of 2%
succinic acid to the third tube. After cutting off the tip of a 100µL pipette tip, we pipetted 10µL
of the worm solution in the centrifuge tube onto the inner circles of each of 3 Petri dish plates.
Immediately after, we pipetted 2µL of the ethanol/sodium azide mixture onto each of the points
marked “T” and “C.” Then the lids were put on and the plates were turned over. We followed the
same procedure for the next 6 plates, except 3 received 2µL of the diacetyl/sodium azide solution
and the other 3 had 2µL of the succinic acid/sodium acid solution added to the “T” points. Once
we finished preparing the plates, we let them sit in room temperature for an hour before placing
them in the 4ºC fridge to store.

A week later, the plates were removed from the fridge and used a microscope to count the
number of adult worms in each quadrant of every plate. Each plate was scored by following the
equation: (# of worms in T - # of worms in C)/(# of worms counted). For each group of 3 plates
with different chemicals, we averaged the chemotaxis indices and calculated the standard error of
the mean values using Excel. An ANOVA was performed on all the individual chemotaxis index
values using SPSS. Upon generating a significant result (p ≤ 0.05), a Tukey Post-Hoc test was
performed using the same software to determine which groups significantly differed.

Results

To test whether succinic acid was a chemoattractant, a chemotaxis assay was performed
with succinic acid, diacetyl, and ethanol for a control. The average chemotaxis index was
calculated for plates with each chemical (Fig. 2). Using SPSS software to run an ANOVA of all
the chemotaxis indices, there was a significant difference (p = 0.003) among the groups. A
Tukey Post-Hoc test revealed a significant difference between both ethanol and diacetyl (p =
0.005) and ethanol and succinic acid (p = 0.004). However, there was no significant difference
between the diacetyl and succinic acid groups (p = 0.969). The significant positive chemotaxis
score indicates succinic acid is a chemoattractant for C. elegans.
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0.7 0.481 0.510


0.6
0.5
Chemotaxis Index (CI)
0.4
0.3
0.2
0.1 -0.153
0
Ethanol Succinic Acid Diacetyl
-0.1
-0.2
-0.3
Chemotactic Agents

Figure 2. The average chemotaxis index for each chemical after a chemotaxis assay with C. elegans.
Ethanol was used as the control, diacetyl was the positive control as the known chemoattractant, and
succinic acid was the test chemical.

Discussion

A calculated chemotaxis index can range between -1 and 1, where values between -0.5
and -1 indicate a chemorepellent, values around 0 suggest no effect, and values between 0.5 and
1 designate a chemoattractant. Based on the results in this experiment, diacetyl was confirmed as
a chemoattractant with a CI greater than 0.5. In addition, the succinic acid was significantly
different from the control in the positive direction and produced chemotaxis score of 0.5 if
rounded, suggesting this chemical as a possible chemoattractant. In the Tukey Post-Hoc test for
between-groups, succinic acid did not significantly differ from diacetyl, and with a similar CI
value, this further implies succinic acid as a chemoattractant. Ethanol produced a negative CI
value close to 0, meaning it is still a good choice for a control because it does not direct
nematode locomotion (Lee, Jee, & McIntire, 2009).

While these results indicate succinic acid is a chemoattractant, the chemical was tested at
a 2% concentration compared to the 1% diacetyl. The reason 2% was tested for succinic acid is
for the time needed to run the chemotaxis assay in class, it was easier and faster to measure the
larger amount of succinic acid necessary for making a 2% solution than a 1%. Even though there
is no literature on succinic acid as a chemoattractant for C. elegans, another chemotaxis assay
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with 1% succinic acid should be conducted in comparison with 1% diacetyl so that the same
concentration of chemicals is reached by all the nematodes in the experiment. It is possible that
the higher concentration of succinic acid is what drew the nematodes towards it more than the
nature of the substance. Upon results pointing to 1% succinic acid as a chemoattractant, then
future work should include what receptor the succinic acid did bind to, whether it was ODR-10
or another one. If succinic acid did bind to the ODR-10, this suggests a similar structure could
produce the same effect as the natural binding chemical. This information could then be utilized
in designing drug treatments that target specific receptors. In all, this work demonstrates the
possibility of crediting structural similarity between succinic acid and diacetyl in producing the
same chemoattractant response from C. elegans that should be further investigated.
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LITERATURE CITED

Bargmann, C.I. Chemosensation in C. elegans (October 25, 2006), WormBook, ed. The C.
elegans Research Community, WormBook, doi/10.1895/wormbook .1.12 3. 1,
http :// www .wormbook.org.

Lee, J., Jee, C., & McIntire, S. L. (2009). Ethanol Preference in C. elegans. Genes Brain Behav., 8(6),
578–585.

Troemel, E. R., Kimmel, B. E., & Bargmann, C. I. (1997). Reprogramming Chemotaxis Responses:
Sensory Neurons Define Olfactory Preferences in C. elegans. Cell, 91(2), 161-169.

URL 1. Succinic Acid. (n.d.). Retrieved April 17, 2018, from


https://thechemco.com/chemical/succinic-acid/

Zhang, Y., Chou, J. H., Bradley, J., Bargmann, C. I., & Zinn, K. (1997). The Caenorhabditis
elegans Seven-transmembrane Protein ODR-10 Functions as an Odorant Receptor in
Mammalian Cells. Proc Natl Acad Sci U S A, 94(22), 12162–12167.